Investigating the role of Runx1 in the specification of haematopoietic stem cells from early precursors in the embryo using a Runx1 reactivatable knockout mouse model

Abstract

Runx1 is a central transcription factor in the development of the murine haematopoietic system and in the emergence and specification of its main key component, the haematopoietic stem cell. Previous studies suggested a requirement for Runx1 in a window of time stretching from mesoderm specification (E6.5) to mid-gestation (E11), but these studies did not investigate each primary haematopoietic site separately. During this PhD project, a Runx1 reactivatable knockout mouse model was used to study the impact of the absence of Runx1 from E9.5 to E11 in primary haematopoietic sites on early precursor populations, especially PreHSC Type I and II. At E9.5, the KO conceptus was already developmentally retarded, lacking progenitors and PreHSC Type II but was not devoid of PreHSC Type I, as demonstrated by flow cytometry, thus suggesting a requirement for Runx1 in the transition from PreHSC Type I to PreHSC Type II stage. Using a novel culture system that enables the potent in vitro maturation of precursors of HSCs into fully mature adult-repopulating HSCs, it was found that maturation of PreHSC Type I into HSCs was hindered in KO tissues, despite the expression of Runx1 in OP9 niche compartment, thus pointing towards a cell autonomous requirement for Runx1. In this model, the Runx1 allele was subsequently reactivated to a functional state by tamoxifen-induced Cre-mediated recombination (CreERT2 system). Tamoxifen / Cre toxicity on HSC maturation was evaluated during AGM reaggregate culture to achieve the best balance between the highest recombination levels and the lowest toxicity when Cre was induced in cell suspension prior to reaggregation. It was found that reactivation of Runx1 at E9.5 in primary haematopoietic sites was not sufficient to rescue haematopoietic development, thus suggesting a requirement for Runx1 before E9.5.